I. Some molecules have hydrogen atoms bound to their structure which—under some conditions—are continuously exchanged with hydrogen atoms present in the surrounding solvent (e.g. water). A typical example of this phenomenon is the hydrogen present at the amide group in the peptide bond. This particular amide hydrogen can be exchanged with other hydrogens present in the surrounding water. One characteristic of this phenomenon is that hydrogens present in peptide bonds more accessible to the water (e.g. those present in the in the surface of the protein) present faster exchange rates than those localized in the inner part of three-dimensional structure of the protein or belong to peptides bond where solvent accessibility is restricted by another protein and/or peptide and/or other portions of its three-dimensional structure. Now, if the solvent in which the protein is dissolved in changed from water (H2O) to deuterium oxide (D2O), the protein will exchange its amide hydrogen for deuterium. Deuterium, also called heavy hydrogen, is a stable isotope of hydrogen. Even though deuterium presents some difference in its physicochemical properties than hydrogen, proteins incorporate deuterium in their peptide bonds without significant disturbances in their structure and function. Because deuterium is heavier than hydrogen, the amount of deuterium incorporation into a polypeptide can be monitored by mass spectrometry. By measuring the amount of deuterium incorporated into the protein (e.g. deuterium incorporated versus incubation time), valuable information can be obtained about for example: protein/protein interactions, protein/drug interactions, protein/peptide interactions, protein/DNA interactions, protein/RNA interactions and peptide/peptide interactions.
When analyzing amide hydrogen exchange samples, some important technical issues need to be considered. Since after the exchange reaction, the sample is normally subjected to liquid chromatography or acidification using solvents containing water (H2O), the sample can easily reverse its deuteration state (the back change of deuterium to hydrogen) if the necessary precautions are taken. Because the exchange reaction is strongly pH dependent, the minimum exchange rate occurs at approximately pH 2.6 (for backbone amide hydrogen—or deuterium—of polypeptides). By performing the exchange at neutral pH and then rapidly drop the pH between 3 or 2, the back exchange rates of the deuterated polypeptides can be dramatically slowed, or quenched.
Hydrogen-deuterium exchange has taken an increasingly important role in drug development. Mapping the conformational changes of a target protein upon the binding of a ligand can accelerate the drug discovery pipeline by giving supplementary data to x-ray crystallography experiments for computational drug design. In addition, since hydrogen-deuterium exchange data gives information about conformational of the protein in solvent—and over time—, it is highly informative when combined with x-ray crystallography.
The following section describes the state-of-the art methodologies to perform hydrogen/deuterium exchange. The section only discusses technical issues relevant to the present invention. A detail explanation of the technique can be found in scientific literature. The aim of this section is to evaluate the differences and advantages of the present invention over the existing technologies. The advantages of the present invention over the state-of-the-art methodologies are explained on the “Description of the Invention” section. The state-of-the-art methodologies can be classified under the following groups:
1. Online Mixing of Target Molecule with Deuterated Solvent.                This system consists in mixing two flow streams using a T-connector. One flow stream carries the sample and the other carries the deuterated solvent (e.g. deuterium oxide). The exchange reaction starts as soon as both streams are mixed. The exchange reaction is analyzed by connecting the outlet of the system to electrospray ionization mass spectrometry and/or collecting the reaction products at the outlet of the system for further analysis. Using this method, the incubation time of the exchange reaction is inversely proportional to the overall flow rate of the system, thus deuteration level versus time graphs can be obtained using different flow rates. The main disadvantage of this approach is the necessary dilution of the sample in the deuterated solvent, since about 90 percent of total volume of reacting solution should correspond to the deuterated solvent. The later is an important problem, since the necessary dilution forces the user to utilize a high amount of sample for each analysis.        
2. In-Tube Mixing of Target Molecule with Deuterated Solvent Followed by Reverse-Phase Liquid chromatography.                The sample is mixed with a deuterated solvent (e.g. deuterium oxide), and aliquots of the reaction are taken over time followed by online or off line mass spectrometry (e.g. 10 aliquots over a time period from 0 to 3 hrs). The laborious and meticulous nature of the sample-handling (in order to avoid back exchange) is the mayor drawback of this approach. Immediately after collection, the samples need to be mixed with acid (e.g. trifluoroacetic acid) in order to drop the pH to levels where the back exchange is minimized Additionally, directly after the acidification step samples are dipped in liquid nitrogen to promote rapid freezing to further minimize the back exchange. For analysis, samples are defrosted and quickly analyzed by online LC MS. Besides the labor-intensive characteristics of this technique, each step can potentially add errors to the reaction (e.g. lack of reproducibility in the collection-timing, acidification and froze/defrost procedures).        
3. In-Tube Mixing of Target Molecule with Deuterated Solvent Followed by Pepsin Digestion and Reverse-Phase Liquid Chromatography.                This methodology is similar to the explained in the previous methodology (entitled: “In-tube mixing of target molecule with deuterated solvent followed by reverse-phase liquid chromatography”). In brief, the sample is mixed with a deuterated solvent (e.g. deuterium oxide), and aliquots of the reaction are taken at different times followed by acidification/freezing. After, posterior online pepsin digestion and reverse-phase chromatography (e.g. 10 aliquots over a time period from the beginning of the experiment to 3 hrs). This technique shares the same drawback from the previous paragraph (entitled: “In-tube mixing of target molecule with deuterated solvent followed by reverse-phase liquid chromatography”). Briefly, labor-intensive and potentially prompt to errors due to the many sample-handling steps (e.g. lack of reproducibility in the collection-timing, acidification and froze/defrost procedures).        
Other Considerations.
An important characteristic shared by all the methodologies described above is the following. In order to obtain an efficient deuteration reaction, the target molecule needs to be dissolved in an excess of a given deuterated solvent (e.g. deuterium oxide). Now, since most of target molecules are dissolved in aqueous buffers (or pure water), the necessary dilution of the sample in the deuterated solvent—normally using dilution factors between 4 to 10 times—is detrimental for the overall sensitivity of the technique, requiring the use of a higher amount of sample per experiment.
II. Chemical analysis lies at the heart of modern science. Advances in analytical chemistry provide the scientific community with tools to advance in their respective research field, as from example in the drug development and pharmacokinetics, toxicology, diagnostics, environmental analysis, and in any other field were a chemical analysis is needed.
One powerful method of chemical analysis is mass spectrometry, in which ionized molecules or fragments thereof are analysed in order to obtain their mass-to-charge ratio based on their translational behaviour in an electric, magnetic or electromagnetic field. Various forms of mass spectrometers are known, such as quadrupole MS, quadrupole ion trap MS, time of flight MS, sector MS, and others. Depending on the method of ionization the molecule may undergo different amounts of fragmentation. By detecting ionized molecule or fragments thereof and comparing with databases on known molecules the chemist may be able to discern the identity of the molecule. For certain molecules however, there may be problems in that the fragment distributions may be very similar, so that it is almost impossible to guess at its identity.
In later years new methods of ionizations have been developed increasing the utility of MS. Most notably there has been developed ionization methods that lets the target molecule remain intact. One such method is APCI (atmospheric pressure chemical ionization), in which excited inert molecules are brought in contact with the target compound, so that the excitation energy ionizes the target. Examples of such ionization devices can be found in documents US 2007/0187589, and U.S. Pat. No. 6,949,741 which are hereby incorporated by reference. Another example can be found in “Design and Performance of a New Combination Electro Spray and Atmospheric Pressure Chemical Ionization Source”, Victor V. Laiko and Craig M. Whitehouse, Proceedings of the 56th ASMS Conference on Mass Spectrometry and Allied Topics, Denver Colo., Jun. 1-5, 2008.
III. Molecular interaction studies are important in drug development and diagnostics as well as for the understanding of diseases at the molecular level. Most of the technologies utilized to investigate molecular interactions are based either on labeling of the target molecule (or the ligands) with a fluorescent dye, radioactive or UV/visible absorbing molecule or on attaching the target molecule to a solid surface, as in surface plasmon resonance or quartz crystal microbalance. The necessary modification of the target molecule (or ligand) and the blockade of the structure of the target molecule (or ligand) involved in linking it to the solid surface are the main problems of these technologies. First of all, the chemical modification of the target molecule (or ligand) has the potential to modify the interaction by giving false-positive binding, decreased or weaker binding or by totally suppressing the binding of the ligand. Also, the hiding or blocking of the molecular structures of the target molecule (or ligand) that are associated with the linkage onto the solid support has the potential to interfere with the normal interaction of the target molecule with the ligand.
Combinatorial chemistry is a technique by which large numbers of structurally distinct molecules may be synthesized at a time. The key of combinatorial chemistry is that a large range of analogues is synthesised using the same reaction conditions, in the same reaction vessels. In this way, the chemist can synthesise many hundreds or thousands of compounds in one time instead of preparing only a few by simple methodology. In its modern form, combinatorial chemistry has probably had its biggest impact in the pharmaceutical industry. Researchers attempting to optimize the activity profile of a compound create a “library” of many different but related compounds. Advances in robotics have led to an industrial approach to combinatorial synthesis, enabling companies to routinely produce over 100,000 new and unique compounds per year.
A challenge to the pharmaceutical industry is how to analyze such libraries comprising a vast number of compounds. Their potential interactions with target molecules of interest need to be analyzed as accurately and as early as possible in the research process.